Sandwich assay kits for detecting shigatoxin ctx3c

ABSTRACT

A sandwich immunoassay kit for detecting ciguatoxins based on a combination of two anti-ciguatoxin CTX3C monoclonal antibodies produced by hybridomas, 3D11 (deposited at IPOD, AIST under accession number FMRM PB-8293) and 10C9 (FMRM PB-8292). In particular, one of the antibodies is labeled and each of them binds specifically to a different site of ciguatoxin CTX3C.

FIELD OF THE INVENTION

The present invention relates to sandwich immunoassay kits, which areuseful for detecting the large non-protein molecule, in particular,ciguatoxins, featuring a use of at least two kinds of monoclonalantibodies, which comprise at least labeled monoclonal antibody, such asenzyme labeled monoclonal antibody and at least one non-labeledmonoclonal antibody. Each of them binds specifically to a different partof ciguatoxins to be detected, and was prepared by using a synthetichapten derived from a different partial structure of ciguatoxin CTX3C.

Further, the present invention relates to hybridoma 3D11 deposited underaccession number FMRM PB-8293 producing the monoclonal antibodydescribed above. A protein conjugate with the synthetic hapten was usedas the antigen for preparation of the hybridoma 3D11. The presentinvention also relates to hybridoma 10C9 deposited under accessionnumber FMRM PB-8292 producing the monoclonal antibody described above. Aprotein conjugate with the different synthetic hapten was used as anantigen for the preparation of the hybridoma 10C9.

BACKGROUND OF THE INVENTION

The investigation of marine toxins by immunoassay, in particularciguatoxins, was initiated from around 1977 along with the developmentof radioimmunoassay. Ciguatera poisons can be collected in a traceamounts from nature. For example, from 850 moray eels, namely from 4tons of moray eels, only 0.35 mg of main poisonous constituentciguatoxin is collected. And also, since it is difficult to produceciguatoxin by cultivation, production of antibodies against ciguatoxinshas been hampered. Hokama et al. of Hawaii University prepared a proteinconjugate by coupling of ciguatoxin (1 μg) with human serum albumin bycarbodiimido method and immunized on mice with said conjugate as anantigen, and elicited a monoclonal antibody (Toxicon Vol. 15, 1977, page317) that bound to ciguatoxin. However, the antibody exhibited strongcross-reactivity with okadaic acid, and the difference of affinity isonly about 5 times (Journal of Clinical Laboratory analysis Vol. 6,1992, page 54). Furthermore, the antibody showed cross-reactivity withbrevetoxins, maitotoxin or palytoxin (Journal of AOAC International Vol.81, 1998, page 727), however, detailed deta has not been reported. Areagent or a kit (Cigua Check TM) for the detection of fishes pollutedwith ciguatoxins by an immunoassay has been developed utilizing theHokama's antibody.

At the earlier stage, the inventors of the present invention synthesizedABC ring fragment, the left end of ciguatoxin, and prepared three kindsof monoclonal antibodies using protein conjugate obtained by utilizingthe ABC ring fragment as a synthetic hapten, however, these monoclonalantibodies showed very weak affinity to ciguatoxin (Synthesis, 1999,page 1431). Other groups have also examined the immunization of proteinconjugates of synthetic hapten (JKLM ring fragment), however, they havenot succeed to prepare monoclonal antibody yet (Toxicon Vol. 38, 2000,page 669).

In the above circumstance, the inventors of the present invention havedesigned and synthesized a synthetic hapten derived from IJKLM ringfragment, which is a partial structure of right end of ciguatoxin CTX3C.The inventors also have established a hybridoma (deposited under theaccession number FMRM PB-8293) by immunization of mice with a proteinconjugate of the synthetic hapten and thereby have succeeded to producea monoclonal antibody, which has high specificity to ciguatoxin CTX3C,using said hybridoma. And said monoclonal antibody is named as 3D11[Deposit date at IPOD (International Patent Organism Depositary), AIST(National Institute of Advanced Industrial Science and Technology) wasMar. 5, 2002 and was deposited by request for transference according toBudapest convention on Feb. 13, 2003 under accession number FMRMPB-8293]. Dissociation constant of said monoclonal antibody 3D11 tociguatoxin CTX3C is 122 nm. Further, the cross-reactivity of saidantibody with structurally related polycyclic ether type marine toxinswas investigated. Very weak cross-reactivity of the antibody 3D11 withred-tide toxin brevetoxins was detected, in which the affinity withbrevetoxin was less than one 350^(th) of the Kd value compared to thatwith CTX3C.

Further, as the synthetic hapten which is used in order to obtainmonoclonal antibody of ciguatoxins, conjugate is synthesized byconjugating the derivative prepared by introducing carboxylic acidlinker to C16 site of ABC ring fragment of ciguatoxin with BSA or KLHwhich are carrier proteins, emulsified the conjugate in RIBI adjuvantand injected the emulsified conjugate to five Balb/c mice for 4 timesand immunized these mice, then spleen is picked out from these mice,cells of said spleen are fused with myeloma cells, P3X63-Ag8.653, andobtain said antibody and reaction specificity of the antibody to thesynthetic hapten is estimated (Synthesis 1999, No. SI. 1431-1436 ISSN0039-7881). That is, using ciguatoxins, in particular, partial structureof ciguatoxin CTX3C, study of synthetic hapten using a part of ringfragment of synthetic ciguatoxin used for the production of monoclonalantibody which is useful for the investigation of ciguatoxin CTX3C byimmunological method is carried out. Concerning above mentioned study,the inventors of the present invention has continued designing asynthetic hapten through which can be obtained monoclonal antibody whoseaffinity to ciguatoxin is further improved. Further, since ciguatoxin isa large molecular whose molecule length is approximately 3 nano meter,ABCDE ring fragment, which is the partial structure of left-handterminus of ciguatoxin, is designed as the hapten and investigated themethod for approach for the synthesis. By the method to immunize a mousewith the protein conjugate of this synthetic hapten, above mentionedhybridoma 10C9 deposited under accession number FERM PB-8292 [Depositdate at the IPOD (International Patent Organism Depositary), AIST(National Institute of Advanced Industrial Science and Technology) wasMar. 5, 2002 and was deposited by request for transference according toBudapest convention on Feb. 13, 2003 under accession number FMRMPB-8292] is prepared, and succeeded to produce above mentioned onemonoclonal antibody which has high specificity to ciguatoxins using saidhybridoma.

The subject of the present invention is basically to provide a kit todetect ciguatoxins by sandwich method whose detective feature is moreimproved by using combination of two kinds of monoclonal antibodies.Further, for the purpose to accomplish said basic subject, the subjectof the present invention is to provide two kinds of hybridomas whichproduce each monoclonal antibody to obtain said kit, two kinds ofsynthetic haptens useful to obtain each hybridoma and synthetichapten-protein conjugates useful to obtain hybridoma which produce saidmonoclonal antibody prepared by combining the synthetic hapten tocarrier protein. As the first step, for the purpose to produce two kindsof monoclonal antibodies used for the sandwich method, two kinds ofnovel compounds used as synthetic haptens, one of them having IJKLM ringof composing ciguatoxin CTX3C and the other having ABCDE ring fragmentof CTX3C are respectively synthesized.

Then, by immunizing with each protein-hapten product obtained byreacting each synthetic hapten with carrier protein, hybridoma whichproduce each monoclonal antibody reacting specifically to ciguatoxinsCTX3C is obtained. And to one of said monoclonal antibody a labelingcompound is bonded and by combining the labeled monoclonal antibody withother non labeled monoclonal antibody, sandwich immunoassay kit whichcan reduce false positive remarkably can be obtained and the technicalconcept of said sandwich immunoassay kit is established.

DISCLOSURE OF THE INVENTION

The first one of the present invention is (1) a sandwich immunoassay kitfor detecting ciguatoxins, comprising of two monoclonal antibodies thatare prepared by using synthetic haptens represented by formula 1 andformula 2, and are produced from hybridoma 3D11 deposited underaccession number FMRM PB-8293 and 10C9 deposited under accession numberFMRM PB-8292, respectively.

in above formulae, R is H or methyl group.

Desirably, (2) the first one of the present invention is a sandwichimmunoassay kit for detecting ciguatoxins of (1), wherein one ofmonoclonal antibodies is labeled, further desirably, (3) the first oneof the present invention is a sandwich immunoassay kit of (2), whereinthe labeling constituent is an enzyme.

(4) The second one of the present invention is a compound represented byabove mentioned formula 1.

(5) The third one of the present invention is an use of the compoundrepresented by formula 1 as a synthetic hapten for the development of ahybridoma that produces a monoclonal antibody against ciguatoxins.

(6) The fourth one of the present invention is a monoclonal antibodyagainst ciguatoxins, which is elicited by immunization of a conjugaterepresented by formula 3 of the synthetic hapten with a carrier protein.

In formula 3, n is an integer of 1 or more.

(7) Desirably, the fourth one of the present invention is the monoclonalantibody of (6) elicited by using protein conjugates of synthetichaptens, wherein the carrier protein is bovine serum albumin, keyholelimpet hemocyanin or egg alblumin

(8) The fifth one of the present invention is hybridoma 3D11 whichproduces monoclonal antibody against ciguatoxins, which is deposited atIPOD of AIST in Japan under accession number FMRM PB-8293.

(9) The sixth one of the present invention is a compound comprising adiastereomer mixture represented by above mentioned formula 2.

(10) The seventh one of the present invention is a reactivefunctionality that is capable of conjugation with a carrier protein,which is required for the development of a hybridoma producing amonoclonal antibody against ciguatoxin CTX3C.”

In formula 4, —O—L is a leaving group for condensation with a carrierprotein.

(11) Desirably, the invention is the novel compound of (10), wherein—O—L is carbodiimide or N-hydroxysuccinimide.

(12) The eighth one of the present invention is protein conjugaterepresented by general formula 1 prepared by condensation of thecompound represented formula 4 with a carrier protein.”,

In the formula, n is a positive integer of 1 or more.

(13) Desirably the invention is the protein conjugate compound of (12),wherein the carrier protein is bovine serum albumin (BSA), keyholelimpet hemocyanin (KLH) or egg albumin (OVA).

(14) The ninth one of the present invention is a monoclonal antibodyagainst ciguatoxin CTX3C elicited by immunization using the proteinconjugate of (13). And,

(15) The tenth one of the present invention is hybridoma 10C9 depositedat IPDO of AIST in Japan under accession number FMRM PB-8292 thatproduces monoclonal antibody against ciguatoxin CTX3C.”

BRIEF ILLUSTRATION OF DRAWINGS

FIG. 1. 1-5 indicate antibody titer of the sera against conjugate AE-BSAprepared by binding A-E ring fragments of ciguatoxin to BSA, (1)-(5)indicate antibody titer of the sera against conjugate IM-BSA prepared bybinding I-M ring fragments of ciguatoxin to BSA.

FIG. 2. The drawing of left hand side shows the inhibition data ofbinding of 10C9 antibody and ABCDE-BSA against adding ABCDE ringfragment (upper) and CTX3C (lower). The drawing of right hand side showsKlotz plotted data of the left drawing, and from the slope of thoselines the binding dissociation constant can be calculated.

FIG. 3. 50 μL/well of PBS solution of 10C9 (4.3 μg/mL) is poured intoELISA Plate (83590) of Coaster Co., Ltd and stood for one night at 4° C.Solution is thrown away and PBS containing 1% skimmed milk (400 μL/well)is added and stand for one hour at room temperature. Solution is thrownaway and after washed by PBS-Tween for three times (200 μL/well),diluted solution of CTX3C (50 μL/well) is added and is stood for onehour. After solution is thrown away, washed by PBS-Tween for 3 times(200 μL/well). PBS-Tween solution of 3D11-HPR (1 μg/mL, 50 μL/well) isadded and stand for one hour at room temperature. Solution is thrownaway and washed by PBS-Tween for 3 times (200 μL/well), then OPDsolution (100 μL/well, Sigma Co., Ltd. FAST (T.M.) o-PHNYLENE DIAMINEDIHYDROCHLORIDE SETS is used) is added and colored for 5-10 minutes atroom temperature. 2N sulfuric acid aqueous solution (50 μL/well) isadded to stop the reaction and absorbance (450 nm) is measured byMicroplate Reader Benchmark of Bio-Rad Co. Ltd. Results are shown.

PREFERRED EMBODYMENT OF THE INVENTION

The present invention will be illustrated more in detail.

A. Establishment of the method of detection for compound which existstrace amount in environment or foods, especially, establishment of themethod for precise and accurate detection is strongly required for thesafety life. Since the establishment of the method of a sandwichimmunoassay for detecting non-protein molecule to be detected usingcombination of plural monoclonal antibodies which binds specifically tothe non-protein molecule to be detected obtained by utilizing pluralsynthetic haptens derived from partial structure in a molecule whichcomposes the non-protein molecule to be detected of the presentinvention provides highly precise detection method, said method can besaid as an innovational technique.

B. Compound 3 contained in synthetic hapten represented by formula 1 ofthe present invention can be obtained by following scheme 1. Thestarting compound 1 for schemata 1 is known (Document; Chem. Comm. 2001,381-382).

Compound 2 is obtained by following process. Compound 1 (7.8 mg, 11μmol), ethylacetate (EtOAc) (100 μL), methanol (300 μL) and Pd(OH)₂(20%, on carbon: 1.1 mg, 11 μmol) are put in a 20 mL eggplant-typeflask. Under the hydrogen atmosphere (thick balloon is used) the contentis stirred for 3 hours at room temperature. After diluted withethylacetate, filtered through celite. Filtered solution is concentratedand the compound 2 (6.2 mg, 13 μmol) is quantitatively obtained.

Mixture of compound 3 and compound 3′ is obtained by following process.Compound 2 (4.2 mg, 9.0 μmol), CH₂Cl₂ (300 μL), (MeO)₂CHCH₂CO₂Me(25 μL,180 μmol) and TsOH.H₂O (0.5 mg, 3 μmol) are put in a 20 mL eggplant-typeflask. After the content is stirred for 1.5 hours at room temperature,toluene (1 mL) is added, vacuumed (120 mbar/hPa) by a rotary evaporatorand put back to atmospheric pressure after 1 hour. Diluted withethylacetate, organic layer is washed with saturated aqueous solution ofsodium hydrogencarbonate, water and saturated brine, dried withmagnesium sulfate anhydride, filtrated and concentrated. Then, it waspurified by silica gel column chromatography and diastereomeric mixtureat acetal position (3:3=3:1) is obtained (4.2 mg, 9.0 μmol, 94%).Property of the mixture of compound 3 and compound 3′ is shown asfollows.

Property of compound 3;

¹H-NMR (500 MHz, CDCl₃) δ 0.89 (3H, d, J=6.5 Hz, Me17), 1.03 (3H, d,J=6.0 Hz, Me16), 1.06 (3H, d, J=7.0 Hz, Me5), 1.15 (3H, d, J=7.5 Hz,Me12), 1.39 (1H, brq, J=11.5 Hz, H9ax), 1.50˜1.62 (4H, m, H17, H6, H4,H16), 1.77-1.88 (4H, m, H20, H19, H5, H4, H6), 1.92-1.97 (2H, m, H19,H20), 2.02 (1H, qdd, J=7.5, 5.0, 3.0 Hz, H12), 2.21 (brdt, J=11.5, 5.0Hz, H9eq), 2.62 (2H, J=5.0 Hz, O₂CHC H₂CO₂Me) 2.85(1H, dd, J=9.5, 5.0Hz, H11), 2.96 (1H, brtd, J=9.5, 3.0 Hz, H7), 3.13 (1H, brddd, J=11.5,9.5, 5.0 Hz, H8), 3.24 (1H, t, J=9.5 Hz, H15), 3.29 (1H, brtd, J=9.5,4.5 Hz, H2), 3.32 (1H, t, J=9.5 Hz, H1), 3.41 (1H, brtd, J=9.5, 3.0 Hz,H3), 3.64 (1H, dd, J=9.5, 1.5 Hz, H14), 3.69 (3H, s, OMe), 3.68-3.72(2H, m, H10, H13), 3.78 (1H, brq, J=7.5 Hz, H21), 3.88 (1H, m, H21),4.01 (1H, dd, J=9.5, 4.5 Hz, H1), 4.87 (1H, t, J=5.0 Hz, O₂CHCH₂CO₂Me).MALDI-TOF MS: Calcd; C₂₉H₄₆O₁₀Na 577.299 (M+Na⁺); Found; 577.244.

C. Preparation process to protein conjugate is shown in scheme 2

Synthesis of compound 3, 3′-compound 4, 4′;

Compound 3, 3′ (4.7 mg, 8.5 μmol), t-BuOH, water (125 μL) and LiOH.H₂O(2.8 mg, 68 μmol) are put in a 20 mL eggplant-type flask, and stirredfor 1 hour at room temperature. After checking pH (3-4 around), dilutedwith ethylacetate (10 mL). Dried with magnesium sulfate anhydride,filtered and concentrated, crude compound of 4, 4′ is obtained. DMF (200μL), N-hydroxysuccinimide (9.7 mg, 85 μmol) and EDC.HCl (8.1 mg, 43μmol) are added and stirred for 12 hours at room temperature. Reactionsolution is diluted with ethylacetate (10 mL), organic layer is washedby water for 3 times, dried with sodium sulfate anhydride, concentratedand compound 6, 6′ which is activated ester is obtained. The solution towhich DMF (100 μL) is added is prepared and used for the preparation ofconjugate.

Preparation Of KLH Conjugate;

To the PBS buffer solution (2.0 mL) of KLH (7.0 mg), DMF solution (50μL) of compound 6, 6′ of activated ester (approximately 4.2 μmol) isadded and stirred for 10 minutes. After standing for one day, thesolution is dialyzed at 4° C. After that, PBS buffer (700 mL) is changedafter 14 and 19 hours, and after 28 hours, transferred from dialysismembrane to Eppendolf tube and preserved at −78° C.

Preparation Of BSA Conjugate

To the PBS buffer solution (2.0 mL) of BSA (7.0 mg), DMF solution (50μL) of compound 6, 6′ of activated ester (approximately 4.2 μmol) isadded and stirred for 10 minutes. After standing for one day, thesolution is dialyzed at 4° C. After that, PBS buffer (700 mL) is changedafter 14 and 19 hours, and after 28 hours, transferred from dialysismembrane to Eppendolf tube and preserved at −78° C.

Pr is carrier protein. N is integer of 1 or more.

Analysis Of Hapten Conjugate;

BSA conjugate obtained by dialysis is analyzed by mass spectrometricanalysis using MALDI-TOF-MS. Average molecular weight of BSA conjugateis approximately 71800 (average molecular weight of BSA is approximately66400). Since molecular weight of hapten is 540, it is understood that10 haptens (mean value of n in compound 6, 6′) are connected to the BSAconjugate in average.

D. Preparation Of Monoclonal Antibody;

To IJKIM-KLH (10 μg) obtained as above, RIBI adjuvant (RIBI Immunol.Product of Res. Inst. Co., Ltd.) is added and stirred well to formemulsion, then said emulsion is given intraperitoneally to Balb/c mice(five mice) 3 times every 2 weeks. On day 35 after the firstimmunization, sera of these mice are collected and antibody titer ofserum is titrated by ELISA method, using IJKLM-BSA, formula 5.

E. Measurement Of Antibody Titer By ELISA Method

50 μL of IJKLM-BSA solution was put into each well of plate for 96 wellELISA (product of FALCON, 3910) and left for 2 hours at roomtemperature, further kept for one night at 4° C., so as for theconjugate to be adsorbed to the plate. The plate was washed by PBS-Tweenfor 3 times [5% Tween 20 (made by Wako Junyaku Co., Ltd.,polyoxyethylene (20) sorbitan monolaurate) or No. 167-11515, made byICI, corresponding to Tween 20 was put into PBS buffer] then washed byMILLI-Q water once and un-adsorbed conjugates was removed. Thesupernatants cultured hybridomas or antiserum, purified antibodysolution were added and left for 1 hour at room temperature, then washedby PBS-Tween and MILLI-Q water. Enzyme labeled secondary antibody (goatanti-mouse IgG-horseradish peroxidase: HRP) (62-6520; product of ZYMED,1000 times diluted) was put into each well, left 1 hour at roomtemperature and washed by PBS-Tween and MILLI-Q water. 100 μL ofsubstrate solution [the contents of the substrate solution: 4.0 mg of1,2-phenylenediamine, 10 μL of hydrogen peroxide aqueous solution, 10 mlof 0.1 M citric acid buffer (pH 5.0) ] was added and processed for colorreaction for 5 minutes, then the reaction was stopped by adding 2Nsulfuric acid (50 μL). Using a micro plate absorbancy measuringapparatus (BIO-RAD, Benchmark 170-6850), absorbance at 450 nm ismeasured and positive clone was judged.

Measurement Of Antibody Titer In Serum;

50 μL of PBS buffer was put into upper most stage of 96-well ELISA platewhich IJKLM-BSA solution was adsorbed. Antiserum of mouse (50 μL)diluted at 200 times was added to the upper most well (A1), then the twofold dilution of this solution was done in order and made from 400 timesto 51200 times dilution in column A1-A8 in tandem. After the plate wasleft for 1 hour at room temperature, and absorbance at 450 nm wasmeasured by above mentioned method. By plotting logarithms of serumdilution rates (abscissa) and absorbance (OD, ordinate), sigmoidtitration curve was obtained, and it became clear that the antibodies inserum bond with IJKLM-BSA conjugate depending on the concentration ofserum. Further, it was confirmed that the antibodies in serum did notbind to ABC-BSA at all.

Among the five mice, the mouse which shows the highest antibody titerwas selected. The selected mouse was supplementary immunizedintraperitoneally with IJKLM-KLH (100 μg) and after 3 days spleen waspicked out from the mouse. Fragments of tissue or organ sticking to thespleen were removed using forceps and the spleen was transferred to apetri dish added basal medium [RPMI Medium 1640 (made by GIBCO, 1 bag),2 g of sodium hydrogencarbonate, 20 g of penicillin-streptomycin (madeby GIBCO) and 20 mL of 200 mM-glutamine were dissolved in distilledwater to prepare a 1000 mL solution with pH 7.2 ) and the cells in thespleen were suspended using forceps. The spleen cells suspended liquidwas filtered and transferred to a 50 mL centrifuge tube and furthermore15 mL of the basal medium was added, pipetted well and filtered toprepare the total volume 30 mL of cell suspension. Centrifuged at 800r.p.m. for 5 minutes at room temperature and supernatant was removed andtapped. 30 mL of HT-BC medium Cmixture of 200 ml of bovine fetus serum(FCS), 20 mL of HT (product of COSMO BIO, HT solution (50 timesconcentrated), 50 mL of BC (Briclone, product of Bioresearch Island) and730 mL of base medium) was added and the cells were suspended.

Preparation Of Hybridoma;

A frozen tube of myeloma cells [P3X63-Ag 8.563 (product of DainihonSeiyaku) ] was took out from the refrigerator (−130° C.) and rapidlydefrosted in a 37° C. incubator. After the tube was sterilized withalcohol cotton, cell suspension in the tube was transferred to 30 mL ofbasal medium. Centrifuged at 800 r.p.m. for 5 minutes at roomtemperature and supernatant was removed then tapped. 10 mL of 10 FCSmedium (prepared by adding 10% FCS to the base medium) was added andprepared the cell suspension, then transferred to a 50 mL culture flask.A cap of the flask was loosen and the flask was placed in CO₂-incubator.Subcultured every 1-2 days and the volume was brought to 2 bottles of250 mL flasks (90- 100 mL).

Spleen cells (2×10⁸ cells) obtained from mouse and myeloma cells (5×10⁷cells) were mixed and centrifuged at 800 r.p.m. for 5 minutes at roomtemperature. Then supernatant was removed and tapped. After that, 30 mLof ECF buffer (prepared by dissolving 45.5 g of mannitol, 10 mM calciumchloride (10 mL), 10 mM magnesium chloride (10 mL) and 20 mM tris buffer<tris(hydroxymethyl)aminomethane> (10 mL) dissolved in distilled waterto make 1000 mL solution with pH 7.2) was added, the supernatant wasremoved and tapped. After these procedures were repeated for 2 times,then ECF buffer (4.8 mL) was added.

1.2 mL each of this solution was pipetted into 6 well plates (made bySUMIRON) and cells was fused using SSH-10 cell fusion apparatus (made byShimazu) by following conditions (distance between electrodes: 1.0 mm,alternating current frequency: 1 MHz, alternating current initial stageapplied voltag: 80V, alternating current initial stage applied time: 10s, pulse duration: 40 μs, pulse voltage: 920V, pulse electric fieldstrength: 2.30 kV/cm, secondary applied alternating current voltage:80V, applied pulse interval: 1.0 s; applied numbers of pulse: 1, pulsevoltage change: +0V, last alternating current applied time: 10 s, ACvoltage damping factor: 0%, contact stregthening; off.

Hybridoma cells prepared above was transferred to 10 plates of 96-wellplate, in which 100 μL of HAT culture medium (selective medium) [themixture composed of 110 mL of base culture medium, 30 mL of FCS, 7.5 mLof BC and HAT (50 times concentrated solution of HAT, which is theproduct of Cosmo Bio Co., Ltd.) ] was contained. After two weeks,hybridomas which produce the antibody that binds to IJKLM ring fragmentof the hapten, were screened using IJKLM-BSA by the ELISA method. Afterpositive wells were selected, and cloned two times, then the positiveclones that were confirmed to produce the antibody in repeated ELIZAwere cultivated successively to proliferate to produce about 200 mL ofthe cell suspension respectively. Consequently, following three kinds ofmonoclonal antibody, whose immunogen was IJKLM-KLM, could be prepared.That was, one was IM (means to have a ring of from I to M) -3D11produces from the hybridoma, which was primary deposited at IPOD(International Patent Organism Depositary) of AIST (National Instituteof Advanced Industrial Science and Technology, in Japan) under accessnumber FERM P-18750 and was transferred by request according to Budapestconvention on February 13, and deposited at IPOD of AIST on Feb. 2, 2003under accession number FERM PB-8293, and the the name of the antidodywas same to the name of the deposited hybridoma, and other ones wereIM-2C7 and IM-8B12. The binding test of the obtained monoclonalantibodies were carried out using ELISA method. As antigens, the BSAconjugate, which were coupled with the partial structure of ciguatoxins,that were ABC, ABCD, A*BC (means A ring fragment is ciguatoxin type) andIJKLM ring structure were used. From the results, as an antibody havingstrong affinity to the IJKLM ring fragment, IgG antibody IM-3D-11 wasselected. Dissociation constant Kd for IJKLM was 8.6 nM (detailsregarding to the method for measurement describe later).

Purification Of Antibody And Determination Of Subclass;

Supernatants were purified using the anti-mouse IgG+IgM affinity columns(product of NFG Industries, Ltd.) [phosphoric acid buffer for bonding(pH 7.0) and buffer for elution (0.2M Glycine-HCl, pH 2.5) ]. Thepurified antibody was confirmed to be>95% in purity by the SDS-PAGEanalysis (sodium dodecyl sulfate-acrylamide gel electrophoresis). Thesubclass of these antibodies were determined using a typing kit (37501)made by PIERCE Co., Ltd.

Analysis Of Affinity Of Antibody (Experiment For CompetitiveInhibition);

Selected three monoclonal antibodies mentioned above, namely IM-3D11,IM-2C7 and IM-8B12, were purified and the dissociation constant (Kd)with hapten was determined. The solution of a serial two-fold dilutionof a competitive inhibitor (each 30 μL of PBS solution) was prepared inplates for ELISA (from A1 to A12 well) in order. Antibody solution (30μL) was added to each plates and left for 2 hours at room temperature.50 μL of the mixed solution of the antibody and the inhibitor was addedto 96-well ELISA plate to which hapten-BSA solution was absorbed (referto affinity analysis of antibody), and left for 20 minutes at roomtemperature. After the plates were washed, absorbance was measured andthe titration curve was obtained. Referring to the method of Friguet etal. [Journal of Immunological Method, vol.77 (1985), page 305) , Kdvalue of the inhibitor was determined from the slope of the straightline obtained by the Klotz plotting. From the results, IM-3D11 was foundto show a high affinity (Kd=8.6 nM) to IJKLM ring fragment (IM) (formula6).

Along with said results, the binding test with toxin's main body CTX3Cwas examined using the above mentioned experimental system forcompetitive inhibition. IM-3D11 bound strongly to CTX3C (Kd=122 nM).Further, as for IM-3D11, the binding test with marine polyether toxinwhose structure is similar was examined, however, bound scarcely withOkadaic acid or Maitotoxin. The cross reactivity with red-tide toxinbrevetoxins was detected, however, compared the affinity to CTX3C, theformer was approximately one 350th (BTXA: Kd=350 μM) and was very weak.

Enzyme Labeling Method Of 3D11 Antibody [Synthesis Of 3D11-HRP(Horseradish Peroxitase) ] (For Sandwich Method);

HRP labeling of 3D11 antibody was carried out by following anexplanatory note of Pierce Co., Ltd., using EZ-Link (T.M.) PlusActivated Peroxitase and kit. 1 mg of aqueous solution (100 μL) ofEZ-Link Plus Activated Peroxide was added to carbonate-bicarbonatebuffer solution (10 mL) of 3D11 antibody (1 mg), and developed thereaction for 1 hour at room temperature. 10 μL of Reductant solution(NaBH₃CN is main reagent) was added to the reaction mixture and left for15 minutes at room temperature. 20 mL of Quench buffer was added and theobtained solution was left for 15 minutes at room temperature. Thereaction solution was dialyzed in 1 LPBS for 3 times, then 1 mL ofglycerol was added and was preserved at −20° C.

As the other labeling components, the known labeling components can beused.

Illustration of the preparation of the monoclonal antibody to becombined with the labeled monoclonal antibody which has ring fragmentA-E (can be abbreviated as AE) of ciguatoxin;

1. Synthesis of diastereomer of above mentioned formula 2 to besynthetic hapten, and mixture of formula 1 and 2 at process 1; Can beobtained by following process 1.

The method for synthesis of the compound of the OH group of compound 7protected with benzyl group (Bn) was disclosed in M. Maruyama et al,Heterocycles, 2001, 54, 93-99. Compound 8 can be obtained bydeprotection of Bn of the compound mentioned in the Document 2 using DDQ(2,3-dichloro-5,6-dicyano- 1,4-benzoquinone)(step 1)), and the alcoholcompound(4.0 mg, 6.0 μmol) obtained in the step1) was dissolved in THF(tetrahydrofuran) (2 mL) and tetrabutylammoniumfluoride (TBAF, 1M THFsolution: 1.8 μL, 18 μmol) was added at room temperature(step 2)). After30 minutes reaction the solution was concentrated, purified by silicagel chromatography. The compound 8 (2.5 mg, 5.9 μmol, 98%) was obtained.

Compound 8 (2.8 mg, 6.6 μmol), CH₂Cl₂ (300 μL), (MeO)₂CHCH₂COOMe (10 μL,70 μmol) and TsOH.H₂O (0.5 mg, 3 μmol) were put in a 20 mL egg planttype flask. After stirred for 1.5 hours at room temperature, toluene (1m) was added and vacuumed by a rotary evaporator (120 mbar/hPa) and putback to the atmospheric pressure after 1 hour. Purified by silica gelchromatography and 1.9 mg (3.8 μmol) of diastereomer mixture regardingacetal site, compound of formula 1 of process 1 : compound of formula2=2:1 was obtained in 57%. The property of mixture of compounds offormula 1 and formula 2 of process 1 are shown below.

The property of mixture of compounds of formula 1 and formula 2 ofprocess 1;

¹H-NMR (500 MHz, CDCl₃) δ 1.53 (1H, m, H10 ax), 2.28 (1H, dt, J=12.0,4.5 Hz, H10 eq), 2.32 (1H, m, H17), 2.37 (1H, m, H4), 2.62 (1H, m, H4),2.64 (1H, m, H17), 2.67 (1H, dd, J=15.5, 5.5 Hz,-H-24), 2.69 (1H,-dd,J=15.5, 5.5 Hz, H24), 3.01 (1H, t, J=9.0 Hz, H8), 3.11 (1H, ddd, J=11.0,9.0, 4.5 Hz, H9), 3.22-3.26 (2H, m, H5, H11), 3.34 (1H, m, H21), 3.43(1H, t, J=10.5 Hz, H22), 3.58-3.64 (3H, m, H16, H7, H6), 3.70 (3H, s,OMe), 3.80 (1H, ddd, J=9.0, 4.0, 2.5 Hz, H12), 4.02 (1H, ddt, J=15.0,4.0, 2.5 Hz, H1), 4.11 (1H, dd, J=10.5, 5.5 Hz, H22), 4.12 (1H, m, H15),4.24 (1H, m, H20), 4.32 (1H, dd, J=15.0, 6.0 Hz, H1), 4.93 (1H, t, J=5.5Hz, H23), 5.63 (1H, brdt, J=12.5, 2.5 Hz, H14), 5.70 (1H, m, H13), 5.73(1H, dd, J=10.5, 5.0 Hz, H19), 5.80 (1H, m, H18), 5.83 (1H, m, H3), 5.92(1H, m, H2).MALDI-TOF MS: Calcd for C₂₆H₃₄O₁₀Na 529.205 (M+Na⁺); Found529.185.

Compound 8 of process 2 was obtained by converting R of compounds offormula 1 and 2 in the process 1 to H by treating the compounds offormulae 1 and 2 with LiOH. The compound shown by the formula 4, whereinL was succinimide, of compound 9 can be obtained by following process 2.

Compound of formula 1 and 2 in the process 1 (2.5 mg, 4.9 μL), t-BuOH(0.5 mL), water (125 μL) and LiOH.H₂O (2.8 mg, 68 mol) were added andstirred for 1 hour at room temperature. KHSO4(18.6 mg, 136 μmol) wasadded and after the pH of solution (3-4 around) was confirmed, dilutedwith ethyl acetate (40 mL). Dried by magnesium sulfate anhydride,filtered and concentrated and crude compound 8 of process 2 wasobtained. To the obtained crude compound 8 of process 2, DMF(DMF=N,N-dimethylformamide, 200 μL), N-hydroxy succinimide (5.6 mg, 4.9μmol) and 4.8 mg (25 μmol) of EDC-HCl(EDC=1-(3-dimethylaminopropyl-3-ethylcarbodiimide) were added andstirred for 12 hours at room temperature. Reaction solution was dilutedwith ethylacetate (40 mL), organic layer was washed by water for 3times. Dried by magnesium sulfate anhydride and concentrated, thencompound 9 of process 2, which was activated ester, was obtained. Thesolution added with DMF (−100 μL) was prepared and used for thepreparation of the conjugate.

2. Preparation Of Protein Conjugate;

Preparation Of KLH conjugate;

To the PBS buffer solution (1.0 mL) of KLH (5.0 mg), DMF solution (50μL) of compound 9 (approximately 2.4 μmol), which was activated ester,was added and stirred for 10 minutes. After left for one day, thesolution was dialyzed at 40° C. PBS buffer (700 mL) was changed after5.9 hours, and after 12 hours transferred from dialysis membrane toEppendolf tube and preserved at −78° C.

Preparation Of BSA Conjugate

To the PBS buffer solution (2.0 mL) of BSA (7.0 mg), DMF solution (50μL) of compound 9 (approximately 2.4 μmol), which was activated ester,was added and stirred for 10 minutes. After left for one day, thesolution was dialyzed at 4° C. PBS buffer (700 mL) is changed after 5.9hours, and after 12 hours transferred from dialysis membrane toEppendolf tube and preserved at −78° C.

3. Analysis Of Haptenic Titer;

BSA conjugate obtained by dialysis was analyzed by mass spectrometricanalysis using MALDI-TOF-MS. Average molecular weight of BSA conjugatewas approximately 70200 (average molecular weight of BSA isapproximately 66400). Since molecular weight of hapten was 476, it wasconfirmed that 8 haptens are connected to the BSA conjugate in average.

4. Preparation Of Monoclonal Antibody (To Be Used As A Non LabeledAntibody);

To ABCDE-KLH (100 μg) obtained as above, RIBI adjuvant (RIBI Immunol.Product of Res. Inst. Co., Ltd.) was added and stirred well so as toform emulsion, then said emulsion was given intraperitoneally to Balb/cmice (five) 3 times every 2 weeks. On day 39 after the firstimmunization, sera of these mice were collected and antibody titers ofthe sera were titrated by the ELISA method using ABCDE-BSA.

ELISA Method;

Into each well of 96-well plates 50 μL of ABCDE-BSA solution was put forELISA (product of FALCON, 3910) and left for 2 hours at roomtemperature, and further left for overnight at 4° C., so as for theconjugate to be adsorbed to the plate. The plate was washed by PBS-Tween3 times [5% Tween 20 (product of Wako Junyaku Co., Ltd., polyoxyethylene(20) sorbitan monolaurate made by the ICI or No. 167-11515 correspondingto Tween 20 ) is contained to PBS buffer], then washed by MILLI-Q wateronce and the un-adsorbed conjugate was removed. The supernatant ofcultured hybridoma or antiserum, purified antibody solution were addedand left for 1 hour at room temperature, then washed by PBS-Tween andMILLI-Q water. Enzyme labeled secondary antibody (goat anti-mouseIgG-West horseradish peroxitase: HRP) (62-6520; made by ZYMED,1000 timesdiluted)was put into each well, left lhour at room temperature andwashed by PBS-Tween and MILLI-Q water. 100 μL of substrate solution[contents of the substrate solution: 4.0 mg of 1,2-phenylenediamine, 10μL of hydrogen peroxide aqueous solution, 10 ml of 0.1M citric acidbuffer (pH 5.0) ] was added and progressed the color reaction for 5minutes, then stopped the reaction by adding 2N sulfuric acid (50 μL).Using a micro plate absorbancy measuring apparatus (BIO-RAD, Benchmark170-6850), absorbance at 450 nm was measured and positive clone wasjudged.

Measurement Of Antibody Titer In Serum;

50 μL of PBS buffer was put in the upper most stage of each 96 wellplate for ELISA to which hapten (ABCDE or IJKLM)-BSA solution wasadsorbed. Antiserum of mouse (50 μL) which was diluted at 200 times wasadded to the well (A1) of the upper most stage ,then the two-folddilution of this solution was done in order (from 400 times to 51200times diluted series were prepared in tandem A1-A8). After the plate wasleft for 1 hour at room temperature, and absorbance at 450 nm wasmeasured by above mentioned method (refer to FIG. 3). By plottinglogarithms of serum dilution rate and absorbance, it was found that theantibodies in serum bound with ABCDE-BSA conjugate depending on theconcentration of serum.

5. Picking The Spleen Out From The Mouse With High Antibody Titer,Cultivation;

The mouse which showed the highest antibody titer was supplementaryimmunized by giving ABCDE-KLH (100 μg) intraperitoneally, and after 3days the spleen was picked out from the mouse. Fragments of tissue ororgan sticking to the spleen are removed by a forceps and the spleen wastransferred to a Petri dish in which basal medium [RPMI Medium 1640(product of GIBCO, 1 bag), 2 g of sodium hydrogencarbonate, 20 g ofpenicillin-streptomycin (product of GIBCO) and 20 mL of 200 mM-glutaminewere dissolved in distilled water to prepare a 100 mL solution with pH7.2 adjusted] was contained and cells in spleen were suspended usingforceps. After the spleen cells suspended liquid was filtered,transferred to a 50 mL centrifuge tube and further 15 mL of basal mediumwas added, pipetted well and filtrated to afford the 30 mL of cellsuspension. Centrifuged at 800 r.p.m. for 5 minutes at room temperatureand supernatant was removed and tapped. 30 mL of HT-BC medium [mixtureof 200 ml of bovine fetus serum (FCS), 20 mL of HT (product of COSMOBIO, HT solution (50 times concentrated), 50 mL of BC (Briclone, productof Bioresearch Island) and 730 mL of basal medium] was added andprepared the cell suspension.

A frozen tube of myeloma cells [P3X63-Ag 8.563 (product of DainihonSeiyaku) ] was took out from a refrigerator (−130° C.) and was thawed inan 37° C. incubator rapidly. After the tube was sterilized with alcoholcotton, the cell suspension in the tube was transferred to 30 mL ofbasal medium. Centrifuged at 800 r.p.m. for 5 minutes at roomtemperature and supernatant was removed then tapped. 10 mL of 10 FCSmedium (prepared by adding 10% FCS to the basal medium) was added andprepared the cell suspension, then transferred to a 50 mL culture flask.The plug of the flask was loosen and was put in a CO₂-incubator. It wassubcultured every 1.2 days, and divided into two 250 mL flasks (90-100mL).

The splenic cells (2×10⁸ cells) took out from mouse and myeloma cells(5×10⁷ cells) were mixed and centrifuged at 800 r.p.m. for 5 minutes atroom temperature. Then supernatant was removed and tapped. After that,30 mL of ECF buffer (prepared by dissolving 45.5 g of mannitol, 10 mMcalcium chloride (10 mL), 10 mM magnesium chloride (10 mL) and 20 mMtris buffer <tris(hydroxymethyl)aminomethane> (10 mL) with pH of 7.2were dissolved in distilled water to make 1000 mL] was added,supernatant was removed and tapped. After these procedures were repeatedfor twice, then ECF buffer (4.8 mL) is added.

1.2 mL each of this solution was pipetted into 6 well plate (made bySUMIRON) and cells were fused by following condition using SSH-10 cellfusion apparatus (product of Shimadzu) (distance between electrodes: 1.0mm, alternating current frequency: 1 MHz, alternating current initialstage applied voltage: 80V, alternating current initial stage appliedtime of: 10 s, pulse duration: 40 μs, pulse voltage: 920V, pulseelectric field strength 2.30 kV/cm, secondary applied alternatingcurrent voltage: 80V, applied pulse interval: 1.0 s; applied number ofpulse: 1, pulse voltage change: +0V, last alternating current appliedtime: 10 s, AC voltage damping factor: 0%, contact strengthening; off].

Hybridoma cells prepared above was transferred to each well of 10 platesof a 96-well plate, in which 100 μL of HAT medium [containing HAT-RPMI1640, 20% FCS, 5% Briclone (made by BioResearch Ireland Co., Ltd.)]After two weeks, hybridomas were screened by ELISA method usingABCDE-BSA. Positive wells were selected, cloned two times, then positivemonoclones that were confirmed to produce the antibody in the repeatedELISA were cultivate successively to proliferate to be about 200 mLrespectively. Consequently, following six kinds of monoclonal antibodyincluding 10C9 (primary deposited at IPOD of AIST under access numberFERM P-18749, on Mar. 5, 2002, and deposited at IPOD of AIST underaccess number FERM PB-8292, by request for transference according toBudapest convention, on Feb. 13, 2003) could be prepared (Table 1).TABLE 1 monoclonal Kd (nM) vs AE Kd (nM) vs antibody hapten CTX3C  1C53.1 3.1  2G5 7.3 23.2  5E6 10.8 19.8  6D5 10.0 16.2 10C6 10.8 26.7 10C90.8 2.8The binding test of the obtained monoclonal antibody was examined byusing ELISA method. As an antigen, BSA conjugate which bound a partialstructure of ciguatoxins was used. From the results of examinations, itwas found that IgG antidody obtained by immunizing ABCDE-KLM ringfragment bound strongly with ABCDE-BSA (chemical formula) and did notbind with IJKLM-BSA (compound IJKLM-BSA).

Evaluation Of The Preparation Of Monoclonal Antibody

Purification Of Antibody And Determination Of Subclass;

The supernatant liquid was purified by using the anti-mouse IgG+IgMaffinity column (made by NGK Industries, Ltd.) [phosphoric acid bufferfor binding (pH 7.0) and a buffer for elusion (0.2M Glycine-HCl, pH 2.5)]. The purified antibody was analyzed by SDS-PAGE (sodium dodecylsulfate-acrylamide gel electrophoresis) and was confirmed that thepurified liquid had >95% in purity. The subclass of each antibodies weredetermined using a typing kit (37501), product of PIERCE Co., Ltd.Analysis of affinity of antibody (experiment for competitiveinhibition); Next, the selected six monoclonal antibodies were purifiedand the dissociation constant (Kd) for hapten was measured. The solutionof serial two-fold dilution of competitive inhibitor (PBS solution, each30 μL) was prepared in the plates for ELISA. The antibody solution (30μL) was added to each plates and left for 2 hours at room temperature.50 μL of the mixed solution of the antibody and the inhibitor was addedto each well of 96-well ELISA plate (product of FALCON, 3910) to whichhapten-BSA solution was adsorbed (0.625 μ/mL), and left for 15 minutesat room temperature. After the plates were washed, the absorbance wasmeasured and the titration curve was obtained. Following the method ofFriguet et al. [Journal of Immunological Method, vol. 77 (1985), page305] , Kd value of the inhibitor was measured and from the slope of thestraight line obtained by Klotz plotting (incline of FIG. 2 b).

From the results, it was found that every six monoclonal antibodies ofTable 1 bound strongly with ABCDE [can be shortened to AE. ABCDE ringfragment (upper compound of compound 6) and CTX3C (lower compound ofcompound 6) ] (Kd<27 nm). Further, it was found that among 6 antibodies,10C9 had the strongest affinity to ABCDE ring fragment (Kd=2.8 nM) andalso to CTX3C (Kd=2.8 nM). Kd values of each monoclonal antibodies wereshown in Table 1.

Further, it was found that the binding affinity of antibody reducesremarkably along with the shortening of ABC ring fragment and molecularsize (compound 7)(Kd value becomes bigger). Results were summarized inTable 2. TABLE 2 monoclonal Kd (nM) vs Kd (nM) vs Kd (nM) vs antibody AEhapten AD hapten AC hapten 1C5 3.1 0.68 42.4 2G5 7.3 0.67 11.1 5E6 10.80.75 53.6 6D5 10.0 0.84 10.0 10C6 10.8 1.1 64.6 10C9 0.8 1.8 73.6

compound 7

Further, the binding test with marine polyether toxin whose structurewas similar was examined, it was found that it did not bind with redtide toxin [Brevetoxin A (BTX A) and Brevetoxin B (BTX B) ], bound withOkadaic acid or Maitotoxin (regarding to the chemical structure of thesecompound, refer to compound 8) scarcely. Results were summarized inTable 3. TABLE 3 monoclonal antibody BTXA BTXB Okadaic acid Maitotoxin 1C5 >100 μM >50 μM >100 μM >25 μM  2G5 >100 μM >50 μM >100 μM >25 μM 5E6 >100 μM >50 μM >100 μM >25 μM  6D5 >100 μM >50 μM >100 μM >25 μM10C6 >100 μM >50 μM >100 μM >25 μM 10C9 >100 μM >50 μM >100 μM >25 μM

Structure Of Polycyclicether Type Marine Toxin

Sandwich Detective Method;

50 μL/well of PBS solution of 10C9 (4.3 μg/mL) was put into ELSA Plate(83590) of Coaster Co., Ltd and left for overnight at 4° C. Solution wasthrown away and PBS containing 1% skimmed milk (400 μL/well) was addedand left for one hour at room temperature. The solution was thrown awayand after washed by PBS-Tween three times (200 μL/well), the dilutedsolution of CTX3C (50 μL/well) was added and left for 1 hour. Aftersolution was thrown away, washed by PBS-Tween three times (200 μL/well).PBS-Tween solution of 3D11-HPR (1 μg/mL, 50 μL/well) was added and leftfor one hour at room temperature. The solution was thrown away andwashed by PBS-Tween three times (200 μL/well), then OPD solution (100μL/well, Sigma Co., Ltd. FAST (T.M.) o-PHNYLENE DIAMINE DIHYDROCHLORIDESETS is used) was added and processed for color reaction for 5-10minutes at room temperature. 2N sulfuric acid aqueous solution (50μL/well) was added so as for the reaction to be stopped and absorbance(450 nm) was measured by Microplate Reader Benchmark of Bio-Rad Co.,Ltd. Measuring results were summarized in FIG. 3.

POSSIBILITY FOR THE INDUSTRIAL USE

As mentioned above, the inventors of the present invention have foundthat ciguatoxins can be detected with a high sensitivity by utilizingSandwich ELISA method using combination of two kinds of monoclonalantibodies which are obtained by using the synthetic hapten having IJKLMring fragment of ciguatoxins and the synthetic hapten having ABCDE ringfragment of ciguatoxins respectively. In the conventional toxindetective kits which use only one antibody having a patial fragment ofciguatoxins capable of binding to the samples, it is impossible to avoidthe result of false positive caused by the antibody bonding withcontamination. In the conventional technique, it is impossible todistinguish whether it is false positive or not, the immunoassay kits ofthe present invention contributes to improve the safety of social liferemarkably. The sandwich immunoassay kit using the combination of twokinds of antibodies to detect non-protein molecule is established by thepresent invention for the first time, and especially, provides excellenteffect from the view point of the establishment of the detective methodwith height specificity to ciguatoxin CTX3C.

Illustration Of The Abbreviations

BSA: bovine serum albumin

CTX: ciguatoxin

ELISA: enzyme linked immunosorbent assay

HRP: Horseradish Peroxidase

O.D.: Optical Density

OPD: o-Phenylene Diamine

PBS: Phosphate Buffered Saline

Bn: benzyl

DDQ: 2,3-dichloro-5,6-dicyano-1,4-benzoquinone

EDC: 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride

FCS: fetal- calf serum

Ig: Immunoglobulin

KLH: keyhole limpet hemocyanine

TBAF: tetra-n-butylammonium fluoride

Ts: p-tolenesulfonyl

THF: tetrahydrofuran

OVA: ovalbumin

1. (canceled)
 2. (canceled)
 3. A sandwich immunoassay kit, comprising oftwo monoclonal antibodies that are prepared by using synthetic haptensrepresented by formula 1 and formula 2, and are produced from hybridoma3D1 1 deposited under accession number FMRM PB-8293 and 10C9 depositedunder accession number FMRM PB-8292, respectively,

wherein, R is H or methyl group.
 4. The sandwich immunoassay kit ofclaim 3, wherein one of the monoclonal antibodies is labeled.
 5. Thesandwich immunoassay kit of claim 4, wherein the labeling constituent isan enzyme.
 6. A compound represented by formula 1


7. (canceled)
 8. A monoclonal antibody against ciguatoxins, which iselicited by immunization of a conjugate represented by formula 3 of thesynthetic hapten with a carrier protein,

wherein, n is a positive integer.
 9. The monoclonal antibody of claim 8elicited by using protein conjugates of synthetic haptens, wherein thecarrier protein is bovine serum albumin, keyhole limpet hemocyanin oregg albumin.
 10. Hybridoma 3D11 that produces the monoclonal antibodyagainst ciguatoxins, which is deposited at IPOD of AIST in Japan underaccession number FERM PB-8293.
 11. A compound comprising adiastereomeric mixture represented by formula 2

wherein, R is H or methyl group.
 12. A compound represented by formula 4having a reactive functionality that is capable of conjugation with acarrier protein, which is required for the development of a hybridomaproducing a monoclonal antibody against ciguatoxin CTX3C,

wherein —O—L is a leaving group for condensation with a carrier protein.13. The compound of claim 12, wherein —O—L is carbodiimide orN-hydroxysuccinimide.
 14. The protein conjugate represented by generalformula 1 prepared by condensation of the compound represented formula 4with a carrier protein,

wherein n is a positive integer.
 15. The protein conjugate of claim 14,wherein the carrier protein is bovine serum-albumin, keyhole limpethemocyanin or egg albumin.
 16. A monoclonal antibody against ciguatoxinCTX3C elicited by immunization using the protein conjugate of claim 15.17. Hybridoma 10C9 deposited at IPOD of AIST in Japan under accessionnumber FERM PB-8292 that produces a monoclonal antibody againstciguatoxin CTX3C.
 18. A detection reagent for ciguatoxins produced byusing a compound represented by formula 1

as the synthetic hapten and the hybridoma which produce the monoclonalantibody to recognize ciguatoxins produced by using the synthetichapten.